It is assumed that the afferent neurotransmitter of cochlear hair cells is glutamate, however, direct evidence is lacking. The specific aim is to test the feasibility of a novel technique for identifying the neurotransmitter release from isolated auditory hair cells. The long-term objectives are to understand the identity of hair cell neurotransmitter and its release mechanism. Our hypothesis is that glutamate is the major neurotransmitter of gerbil inner and outer hair cells. The experimental design is to elicit transmitter release in isolated gerbil inner or outer hair cells by electrically depolarizing membrane potentials. The evoked transmitter release will be detected by outside-out membrane patches containing single or multiple glutamate-receptors excised from cultured postnatal gerbil cortical neurons. In the second stage of the experiments, transmitter release in hair cells will be induced by mechanical deflection to stereocilia by a water jet device. The methods to be used are 1) long- term culture of postnatal gerbil cortical neurons. 2) short-term co- culture of isolated gerbil inner or outer hair cells with postnatal gerbil cortical neurons. 3) obtaining outside-out glutamate-receptor patches from cultured neurons and moving the patch to probe the presence of the glutamate at various locations near the hair cell stimulated to release its transmitter. The positive detection of the glutamate release from hair cells will serve as direct evidence that glutamate is the major neurotransmitter of auditory hair cells. The proposed technique to detect neurotransmitter release is highly sensitive and selective. It could provide a new approach for studying the mechanism of hair cell neurotransmitter release at the cellular level. Successful establishment of the new technique put forth in this proposal may also provide an approach to studying whether regenerated hair cells have normal transmitter releasing functions.